The overall focus of our laboratory is the study of the 70-kDa heat shock proteins and their role in both normal cellular processes and heat shock. First, we are investigating one of the only defined functions of a 70-kDa heat shock protein--the ability of the 70-kDa uncoating (UC) ATPase isolated from bovine brain to remove clathrin from clathrin coated vesicles in an ATP dependent reaction. We previously found that, when the UC ATPase with bound ATP is mixed with coated vesicles, there is an initial burst of uncoating followed by slow steady-state uncoating. Based on these data we proposed a simple model where the UC ATPase has only one binding site for ATP and one binding site for clathrin. This contrasts with a more complicated model proposed by Schmid and Rothman where the UC ATPase has two sites for clathrin and two sites for ATP. We have now obtained data which show that the UC ATPase, indeed, has only one site for clathrin and one site for ATP. Our data also show that both ATP and ADP bind very strongly to the UC ATPase (Kbinding ca= 1 x 108) so that the UC ATPase, as it is normally prepared contains ca one bound ADP per molecule which is very difficult to remove. However removal of this bound ADP does not denature the UC ATPase. We have also studied the ATPase activity of the UC ATPase. In the absence of substrate the UC ATPase shows no initial burst of ATP hydrolysis. However, in the presence of the assembly proteins which are required to polymerize clathrin on coated vesicles, a very rapid initial burst of ATP hydrolysis is observed. In contrast, clathrin does not cause an initial burst of ATP hydrolysis. These data suggest that the assembly proteins are a very specific substrate of the UC ATPase and may be important in the uncoating process. In addition to these enzymatic studies, we have cloned the bovine brain UC ATPase and have expressed it in yeast. We previously showed that three yeast 70 kDa proteins, at least one of which must be present for yeast viability, show a functional similarity to the bovine brain UC ATPase. Preliminary experiments now suggest that the bovine brain UC ATPase can substitute for these three yeast 70 kDa proteins in keeping the yeast viable.